Method for modulating process mediated by farnesoid activated receptors

ABSTRACT

Farnesyl pyrophosphate, the metabolically active form of farnesol, is a key precursor in the synthesis of cholesterol, carotenoids, steroid hormones, bile acids and other molecules involved in cellular growth and metabolism. A nuclear receptor has been identified that is transcriptionally activated by farnesol and related molecules. This novel signaling pathway can be modulated by the use of key metabolic intermediates (or analogs and/or derivatives thereof) as transcriptional regulatory signals.

RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 10/155,379, filed May 24, 2002, now issued as U.S. Pat. No.7,041,498, which is a Divisional Application of U.S. application Ser.No. 09/696,443, filed Oct. 24, 2000, now issued as U.S. Pat. No.6,416,957, which is a Divisional Application of U.S. application Ser.No. 09/469,721, filed Dec. 21, 1999, now issued as U.S. Pat. No.6,184,353, which is a Divisional Application of U.S. application Ser.No. 08/372,183, filed Jan. 13, 1995, now issued as U.S. Pat. No.6,005,086.

FIELD OF THE INVENTION

The present invention relates to intracellular receptors, and ligandstherefor. In a particular aspect, the present invention relates tomethods for selectively modulating processes mediated by farnesoidactivated receptors.

BACKGROUND OF THE INVENTION

Molecular cloning studies have demonstrated that receptors for steroids,retinoids, vitamin D and thyroid hormones comprise a superfamily ofregulatory proteins that are structurally and functionally related (seeEvans, in Science 240:889-895 (1988)). Known as nuclear receptors, theseproteins bind to cis-acting elements in the promoters of their targetgenes and modulate gene expression in response to ligand therefor, suchas a hormone.

Nuclear receptors can be classified based on their DNA bindingproperties (see Evans, supra and Glass, in Endocr. Rev. 15:391-407(1994)). For example, the glucocorticoid, estrogen, androgen, progestinand mineralocorticoid receptors bind as homodimers to hormone responseelements (HREs) organized as inverted repeats (IRs, see Glass, supra). Asecond class of receptors, including those activated by retinoic acid,thyroid hormone, vitamin D₃, fatty acids/peroxisome proliferators andecdysone, bind to HREs as heterodimers with a common partner, theretinoid X receptor (i.e., RXR, also known as the 9-cis retinoic acidreceptor; see, for example, Levin et al., in Nature 355:359-361 (1992)and Heyman et al., in Cell 68:397-406 (1992)).

An important advance in the characterization of the nuclear receptorsuperfamily of regulatory proteins has been the delineation of a growingnumber of gene products which possess the structural features of nuclearreceptors, but which lack known ligands. Accordingly, such receptors arereferred to as orphan receptors. The search for activators for orphanreceptors has created exciting areas of research on previously unknownsignaling pathways (see, for example, Levin et al., (1992), supra andHeyman et al., (1992), supra). Indeed, the ability to identify novelregulatory systems has important implications in physiology as well ashuman disease and methods for the treatment thereof.

Since receptors have been identified for all known nuclear-actinghormones, a question arises as to the types of molecules that mayactivate orphan receptors. In view of the fact that products ofintermediary metabolism act as transcriptional regulators in bacteriaand yeast, such molecules may serve similar functions in higherorganisms (see, for example, Tomkins, in Science 189:760-763 (1975) andO'Malley, in Endocrinology 125:1119-1120 (1989)). For example, a crucialbiosynthetic pathway in higher eucaryotes is the mevalonate pathway (seeFIG. 1), which leads to the synthesis of cholesterol, bile acids,porphyrin, dolichol, ubiquinone, carotenoids, retinoids, vitamin D,steroid hormones and farnesylated proteins.

Farnesyl pyrophosphate (FPP), the metabolically active form of farnesol,represents the last precursor common to all branches of the mevalonatepathway (see FIG. 1). As a result, FPP is required for such fundamentalbiological processes as membrane biosynthesis, hormonal regulation,lipid absorption, glycoprotein synthesis, electron transport and cellgrowth (see Goldstein and Brown, in Nature 343:425-430 (1990)). Becauseof the central role of FPP in the production of numerous biologicallyimportant compounds, it is to be expected that its concentration shouldbe closely regulated. This suggests that cells are likely to havedeveloped strategies to sense and respond to changing levels of FPP, orits metabolites. One possible strategy by which cells can accomplish thedesired regulation is to utilize a transcription factor whose activityis specifically regulated by a low molecular weight signaling moleculesuch as an FPP-like molecule. Potential candidates for such means tosense changing levels of FPP, or metabolites thereof, include members ofthe nuclear receptor superfamily, since these proteins are activated bylow molecular weight signaling molecules.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have discovered that anorphan nuclear receptor, referred to as farnesoid activated receptor(i.e., FAR), is activated by farnesol and related molecules. Thus, FARprovides one of the first examples of a vertebrate transcription factorthat is regulated by an intracellular metabolite. These findings suggestthe existence of vertebrate signaling networks that are regulated byproducts of intermediary metabolism.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the mevalonate pathway and details the relationshipbetween FAR-RXR activators (set off in the figure by enclosure in a box)and the other compounds produced by the mevalonate pathway.

FIG. 2 summarizes the relationship among the DNA binding domains of FAR(Cys¹²⁴-Met¹⁸⁹) and other nuclear receptors (i.e., human peroxisomeproliferator activated receptor (PPARα, Genbank L02932); human retinoidX receptor a (RXRα, Genbank X52773); human retinoic acid receptor α(RARα, Genbank X06538); human thyroid hormone receptor α1 (T³Rα, GenbankM24748); human vitamin D receptor (VDR, Genbank J03258); human orphannuclear receptor (MB67, Genbank L29263); rat orphan nuclear receptor(RLd-1, Genbank U11685); and Drosophila ecdysone receptor (EcR, GenbankM74078). Dendograms were created using the PILEUP program (GeneticsComputer Group, version 7.2, University of Wisconsin).

FIG. 3 presents an amino acid sequence comparison between rat FAR andDrosophila EcR. Similarity between the DNA binding and ligand bindingdomains are schematically represented as percent amino acid identity.Amino acid regions comprising each domain are numbered accordingly.

FIG. 4 demonstrates the interaction of FAR and RXR.

FIGS. 5A, 5B and 5C demonstrate the hormonally controlled activity ofthe FAR-RXR complex. In FIG. 5A, the response of FAR alone, RXR aloneand FAR+RXR to exposure to juvenile hormone III (JH III) is illustrated.

FIG. 5B illustrates the response of RXR alone, thyroid hormone receptor(T₃R) alone, RAR alone and ecdysone receptor+ultraspiracle (EcR+USP) toexposure to ligands selective for each respective receptor species(i.e., 100 nM T₃ (L-triiodothyronine), 1 μM trans-RA (all-trans-retinoicacid) or 100 nM muristerone A, respectively), or to JH III.

FIG. 5C illustrates the response of FAR alone, RXR alone and FAR+RXR toexposure to an FAR ligand (JH III), an RXR ligand (LG69, i.e.,(4-{1-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-napthalenyl)-1-propenyl}benzoicacid), or a combination of JH III and LG69.

FIG. 6A summarizes FAR-RXR activity when exposed to various isoprenoids.

FIG. 6B presents a dose-response profile for exposure of FAR-RXR complexto JH III and farnesol.

FIG. 7 is an abbreviated genetic map showing the localization of the Fxrgene on mouse Chr 10.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a novel member of the nuclearreceptor superfamily has been identified that forms heterodimers withRXR. The resulting FAR-RXR heterodimer complex is activated by farnesoland related metabolites. This FAR-RXR heterodimer binds to ecdysone-likeresponse elements organized as an inverted repeat spaced by 1 nucleotide(referred to herein as IR1), a property that is unique among vertebratenuclear receptors.

Thus, as described in greater detail in the Examples which follow, adegenerate 29-mer consensus oligonucleotide corresponding to the highlyconserved P-box/DNA recognition helix (TCEGCK(G/V)FF; SEQ ID NO:1) ofthe nuclear receptor superfamily DNA binding domain (DBD) was used toprobe a λgtll cDNA library derived from mouse hepatoma Hepa-1c1c7 mRNA.Four positive cDNAs were identified from a low-stringency screen of twomillion clones and subjected to nucleotide sequence analysis. Inaddition to cDNAs for the previously described glucocorticoid andthyroid hormone receptors, two clones encoding novel orphan receptorswere obtained. These cDNA clones were about 850 base pairs each andlacked complete coding sequences.

To obtain the complete open reading frame for OR2, a cDNA library fromregenerating rat liver was screened. A 2.1 kb cDNA was cloned whichencodes a 469 amino acid open reading frame (SEQ ID NO:2). In vitrotranslation of OR2.8 derived RNA results in a protein with a relativemolecular mass (M_(r)) of 54,000, close to the predicted M_(r) of54,135. The OR2.8 cDNA contains a short interspersed repetitive DNAelement (see Sutcliffe et al., in Science 225:1308-1315 (1984)) in the3′ untranslated region, followed by a polyadenylation signal. Asdescribed in detail herein, the OR2.8 cDNA encodes a novel member of thenuclear receptor superfamily that is activated by farnesoids.Accordingly, this novel receptor protein is referred to herein as FAR(Farnesoid Activated Receptor).

Examination of the amino acid sequence of FAR confirms that it is amember of the nuclear receptor superfamily. The region spanningCys¹²⁴-Met²⁸⁹ contains several invariant amino acids, including 4cysteine residues that are characteristic of the DNA binding domain(DBD) of all nuclear hormone receptors. The dendogram in FIG. 2illustrates the relationship of this region to the DBD of otherreceptors. The FAR DBD is most similar to the DBD of the insect ecdysonereceptor (EcR). These receptors share 81% amino acid sequence identitywithin their DBDs (see FIG. 3). The FAR DBD is more distantly related toother members of the nuclear receptor superfamily (see FIG. 2).

The carboxy-terminal ligand binding domain (LBD) of nuclear receptors isa complex region encoding subdomains for ligand binding, dimerizationand transcriptional activation. Analysis of the carboxy terminal regionin FAR (spanning Leu²⁵⁰-Gln⁴⁶⁹) indicates that it possesses only 33%sequence identity (59% similarity) with the corresponding region of theecdysone receptor (see FIG. 3). Within this region, significantsimilarity is confined to regions involved in receptor dimerization (seeForman and Samuels, in Mol. Endocrinol. 4:1293-1301 (1990)), includingthe T_(i) subdomain (48% identity), heptad repeats 4-6 (50% identity)and heptad 9 (75% identity). In addition, the last 22 amino acids, whichpossess transcriptional activation functions in other receptors (seeDanielian et al., in EMBO J. 11:1025-1033 (1992)), are 42% identicalamong FAR and EcR (see FIG. 3). These structural similarities indicatethat FAR is a member of the nuclear receptor superfamily with potentialfunctional relatedness to the EcR.

As used herein, the phrase amino acid sequence similarity refers tosequences which have amino acid substitutions which do not change theinherent chemical properties of the subject polypeptide. Thus, aminoacid sequences wherein an acidic residue is replaced with another acidicresidue, or wherein a basic residue is replaced with another basicresidue, or wherein a neutral residue is replaced with another neutralresidue, retain a high degree of similarity with respect to the originalsequence, notwithstanding the fact that the sequences are no longeridentical.

The ability to respond to metabolic intermediates distinguishes FAR fromother nuclear receptors. FPP, the metabolically active form of farnesol,is a key metabolic precursor in the synthesis of numerous biologicallyactive molecules including proteins (see FIG. 1 and Goldstein and Brown,in Nature 343:425-430 (1990).

Transcriptional regulation by intermediary metabolites such ascarbohydrates, amino acids and lipids is a common paradigm in bacteriaand yeast (see, for example, Sze et al., in Science 258:1143-1145(1992)). In these systems the metabolite, or a related compound, oftenserves as an effector in a transcriptionally-regulated feedback loopthat maintains appropriate concentrations of the metabolite/effector.The demonstration that FAR-RXR is regulated by farnesoid-relatedmetabolites provides an example of this type of regulation invertebrates.

Since farnesoid metabolites are synthesized intracellularly, individualcells which express FAR may also be producing the ultimate FARactivator. Other examples of transcriptional signaling by intracellularmetabolites include the PPAR, a fatty acid-activated orphan receptor(see Gottlicher et al., in Proc. Natl. Acad. Sci. USA 89:4653-4657(1992)) that regulates genes involved in fatty acid metabolism (seeGreen and Wahli, in Mol. Cell Endocrinol. 100:149-153 (1994)) andadipocyte differentiation (see Tontonoz et al., in Genes Dev.8:1224-1234 (1994)). Similarly, the low density lipoprotein receptorgene regulator, SREBP-1, is maintained in an inactive form byhydroxycholesterol (see Wang et al., in Cell 77:53-62 (1994)). Together,these systems define a novel paradigm of metabolite-controlledintracellular (metacrine) signaling in vertebrates (see O'Malley, inEndocrinology 125:1119-1120 (1989). Metacrine signaling provides a meansto regulate responses to intracellular metabolites in a cell-autonomousfashion. By transducing metabolic cues into genomic responses, FAR, PPARand SREBP-1 provide examples of a metabolic code proposed by GordonTomkins in 1975 (see Tomkins, in Science 189:760-763 (1975)).

Activation of classical nuclear receptors occurs at physiologicalconcentrations of circulating hormones, typically in the nanomolarrange. However, the activation of PPAR by naturally occurring fattyacids requires 10-100 μM doses, consistent with the presumedintracellular concentration of these compounds. Physiologicconcentrations of farnesoids have been difficult to determine due totheir rapid metabolism and potential sequestration by intracellular andextracellular binding proteins.

Intracellular concentrations of farnesoids can be inferred from theMichaelis constant (K_(m)) of enzymes that utilize isoprenoidsubstrates. The K_(m) of farnesyl:protein transferases for FPP rangesbetween 0.5 and 8.5 μM (see Gomez et al., in Biochem. J. 289:25-31(1993) and Reiss et al., in Cell 62:81-88 (1990)) and half-maximalinhibition of isopentenyl pyrophosphate isomerase occurs with 10 μM FFP(see Rilling and Chayet, In: Sterols and Bile Acids, eds. Danielsson andSjovall (Elsevier Science; 1985)). Furthermore, several biologicaleffects of JH III and isoprenoids have been reported to occur in the10-100 μM concentration range. For example, induction of ornithinedecarboxylase by phorbol esters and phytohemagglutinin can beantagonized by 100 μM JH III in bovine lymphocytes (see Kensler et al.,in Cancer Res. 38:2896-2899 (1978)). Similarly, down-regulation ofHMG-CoA reductase activity by a mevalonate-derived non-sterol occurswhen mevalonate is added to cells at concentrations in excess of 100 μM(see, for example, Brown and Goldstein, in J. Lipid Res. 21:505-517(1980), and Nakanishi et al., in J. Biol. Chem. 263:8929-8937 (1988)).Moreover, FAR is expressed in the liver, intestine, adrenal gland andkidney: tissues known to support high flux through the mevalonatepathway. Thus, activation of FAR is likely to occur at appropriatefarnesoid concentrations in physiologically relevant tissues.

FPP is known to regulate cell growth by virtue of its ability to alterthe intracellular localization of ras and other proteins via covalentfarnesylation (Goldstein and Brown, Nature 343:425-430 (1990)). Theresults presented herein suggest that in addition to this pathway,farnesoids are also capable of promoting biological changes through anovel transcriptional signaling pathway. Indeed, the identification of afarnesoid-dependent transcription factor provides the opportunity tomodulate a key pathway responsible for the generation of lipids.Furthermore, the initial identification of a farnesoid-dependenttranscription factor suggests that a network of farnesoid-responsivegenes exist. Such genes can readily be identified by suitable meanshaving the detailed information concerning FAR provided herein.

In accordance with the present invention, there is provided a method formodulating process(es) mediated by farnesoid activated receptorpolypeptides, said method comprising conducting said process(es) in thepresence of at least one farnesoid.

Farnesoid activated receptor polypeptides contemplated for use in thepractice of the present invention can be characterized by reference tothe unique tissue distribution thereof. Thus, expression of FARpolypeptides is restricted to the liver, gut, adrenal gland and kidney,all tissues known to have a significant flux through the mevalonatepathway.

Alternatively, farnesoid activated receptor polypeptides contemplatedfor use in the practice of the present invention can be characterizedby:

-   -   (1) being responsive to the presence of farnesoid(s) to regulate        the transcription of associated gene(s);    -   (2) having a relative molecular mass of about 54,000; and    -   (3) having a DNA binding domain of about 66 amino acids with 9        Cys residues, wherein said DNA binding domain has:        -   (a) about 81% amino acid identity with the DNA binding            domain of the Drosophila ecdysone receptor,        -   (b) about 56% amino acid identity with the DNA binding            domain of VDR, and        -   (c) about 45% amino acid identity with the DNA binding            domain of hGR.

Farnesoid activated receptor polypeptides contemplated for use in thepractice of the present invention can be further characterized by:

-   -   (4) having a ligand binding domain of about 220 amino acids,        wherein said ligand binding domain has:        -   (a) about 33% amino acid identity, and about 59% amino acid            similarity, with the ligand binding domain of the Drosophila            ecdysone receptor,        -   (b) about 32% amino acid identity with the ligand binding            domain of VDR, and        -   (c) about 26% amino acid identity with the ligand binding            domain of hGR.

Presently preferred farnesoid activated receptor polypeptidescontemplated for use in the practice of the present invention can becharacterized as having substantially the same amino acid sequence asthat shown in SEQ ID NO:2. Especially preferred farnesoid activatedreceptor polypeptides contemplated for use in the practice of thepresent invention are those which have the same amino acid sequence asthat shown in SEQ ID NO:2.

The phrase “substantially the same” is used herein in reference to aminoacid sequences that have slight and non-consequential sequencevariations from the actual sequences disclosed herein. Species which are“substantially the same as the reference sequence are considered to beequivalent to the disclosed sequences and as such are within the scopeof the appended claims.

Farnesoid compounds contemplated for use in the practice of the presentinvention include compounds having the structure:

wherein

-   -   each R is independently lower alkyl or alkoxy,    -   each R′ is independently selected from hydrogen, lower alkyl or        alkoxy,    -   each R″ is independently selected from hydrogen, lower alkyl or        alkoxy,    -   X is selected from —CH₂OH, —CH₂OAc, —CO₂H, or —CO₂Me,    -   n is 2 or 3,    -   each q is independently 1 or 2,    -   each q′ is independently 1 or 2, and    -   q and q′ are the same.

Exemplary farnesoids contemplated for use in the practice of the presentinvention include those wherein:

-   -   (1) each R is methyl, each R′ is hydrogen, each R″ is hydrogen,        X is —CH₂OH, n is 2, and each q and q′ is 1 (i.e., the farnesoid        molecule is polyunsaturated);    -   (2) each R is methyl, each R′ and each R″ is hydrogen, X is        —CO₂H, n is 2, and each q and q′ is 1 (i.e., the farnesoid        molecule is polyunsaturated);    -   (3) the polyene backbone of the farnesoid molecule contains an        epoxide functionality, each R is methyl, each R′ is hydrogen,        each R″ is hydrogen, X is —CH₂Me, n is 2, and each q and q′ is        1;    -   (4) each R is methyl, each R′ is hydrogen, each R″ is hydrogen,        X is —OAc, n is 2, and each q and q′ is 1;    -   (5) each R is methyl, each R′ is hydrogen, each R″ is hydrogen,        X is —CH₂OH, n is 3, and each q and q′ is 1; and the like.

In accordance with another embodiment of the present invention, there isprovided a method of testing a compound for its ability to regulatetranscription-activating effects of a farnesoid activated receptorpolypeptide, said method comprising assaying for reporter protein whencells containing a farnesoid activated receptor polypeptide and reporterconstruct are contacted with said compound;

wherein said reporter construct comprises:

-   -   (a) a promoter that is operable in said cell,    -   (b) a hormone response element, and    -   (c) DNA encoding a reporter protein,    -   wherein said reporter protein-encoding DNA segment is        operatively linked to said promoter for transcription of said        DNA segment, and    -   wherein said promoter is operatively linked to said hormone        response element for activation thereof.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 Cloning of FAR

A degenerate 29-mer consensus oligonucleotide (5′-ACC TGT GAG GGC TGCAAR GKY TTC TTC AA-3′; SEQ ID NO:3), corresponding to the highlyconserved P-box/DNA recognition helix (TCEGCK(G/V)FF; SEQ ID NO:1) ofthe nuclear receptor superfamily DNA binding domain (DBD) was used toprobe a λgtll mouse hepatoma Hepa-1c1c7 cDNA library of 2×10⁶ clonesunder low stringency conditions (see Issemann and Green, in Nature347:645-650 (1990)). An incomplete 850 bp mouse OR2 cDNA clone wasobtained. This clone was used subsequently to screen a regenerated ratliver cDNA library.

A full length clone (referred to as OR2.8) was obtained from this screenand sequenced by the dideoxy sequencing method. The deduced amino acidsequence thereof is presented herein as SEQ ID NO:2.

EXAMPLE 2 Formation of FAR-RXR Complexes

In order to explore the functional properties of FR, the DNA bindingproperties of this orphan receptor were analyzed. It has previously beenshown that RXR is a common heterodimeric partner required for highaffinity DNA binding by several nuclear receptors (see, for example,Hallenbeck et al., in Proc. Natl. Acad. Sci. USA 89:5572-5576 (1992);Kliewer et al., in Nature 355:446-449 (1992); Leid et al. , in Cell68:377-395 (1992); Marks et al., in EMBO J. 11:1419-1435 (1992); Yu etal., in Cell 67:1251-1266 (1991); and Zhang et al,. in Nature355:441-446 (1992). Moreover, it has been shown that the DNA and ligandbinding activities of the Drosophila ecdysone receptor (EcR) requireheterodimer formation with RXR or USP (the Drosophila homologue of RXR;see O'Malley in Endocrinology 12S:1119-1120 (1989)). As illustrated inFIG. 3, FAR and EcR possess striking similarity within the dimerizationsubdomain of the ligand binding domain (LBD). Furthermore, FAR iscolocalized with sites of RXRα and RXRβ expression (see Example 6below). These observations prompted an investigation as to whether FARcould interact with RXR, or with other members of the nuclear receptorsuperfamily. To do so, a two-hybrid system modified for use in mammaliancells was employed (see, for example, Nagpal et al., in EMBO J.12:2349-2360 (1993)).

Thus, CV-1 cells were transiently transfected (as indicated in FIG. 4)with cytomegalovirus promoter driven expression vectors containing theyeast GAL4 DNA binding domain (DBD) alone (GAL4₁₋₁₄₇), GAL4 linked tothe FAR ligand binding domain (LBD; i.e., GAL4-FAR₁₈₄₋₄₆₉), and the 78amino acid Herpes virus VP16 transactivation domain (VP) linked to theamino terminal end of the LBDs for human RXRα (VP-RXR₂₀₃₋₄₆₂), mousePPARα (VP-PPAR₁₅₅₋₄₆₈), VDR (VP-VDR₉₂₋₄₂₇), T₃Rβ (VP-T₃Rβ₁₇₃₋₄₅₆) orRARα (VP-RAR₁₅₆₋₄₆₂). All cells were cotransfected with a luciferasereporter construct containing 4 copies of the yeast GAL4 upstreamactivating sequence and a β-galactosidase expression vector as internalcontrol.

Thus, CV-1 cells were grown in DMEM supplemented with 10% AG1-X8resin-charcoal stripped calf bovine serum, 50 U/ml penicillin G and 50μg/ml streptomycin sulfate (DMEM-CBS) at 37° C. in 5% CO₂. One day priorto transfection, cells were plated to 50-80% confluence using phenol-redfree DMEM with 10% resin charcoal stripped fetal bovine serum(DMEM-FBS). Cells were transfected (with reporter construct (300 ng/10⁵cells), cytomegalovirus driven receptor (100 ng/10⁵ cells) andβ-galactosidase expression vectors (500 ng/10⁵ cells) as indicated inFIG. 4) by lipofection usingN-{2-(2,3)-dioleoyloxy)propyl-N,N,N-trimethyl ammonium methyl sulfate}according to the manufacturer's instructions (DOTAP, BoehringerMannheim). After 2 hours the liposomes were removed and cells treatedfor 40 hours with phenol-red free DMEM-FBS containing farnesol as theligand. Cells were harvested and assayed for luciferase andβ-galactosidase activity. All points were performed in triplicate andvaried by less than 10%. Experiments were repeated three or more timewith similar results. Data points were normalized for differences intransfection efficiency using β-galactosidase, and plotted as relativeactivity where the untreated reporter is defined to have an activity of1 unit.

As seen in FIG. 4, neither the GAL4 DBD, nor the GAL4-FAR chimera arecapable of stimulating transcription from a reporter constructcontaining the GAL4 upstream activating sequence. Similarly, a fusionprotein containing the Herpes virus VP16 transactivation domain linkedto the RXRα-LBD (VP-RXR) is inactive when expressed alone or with theGAL4 DBD. However, when GAL4-FAR and VP-RXR are coexpressed, thereporter is dramatically activated (by about 500-fold), indicating thatFAR and RXRα interact efficiently in cells. Using similar VP16-LBDfusion proteins, no interaction could be detected between FAR andreceptors for peroxisome proliferators/fatty acids (PPAR), vitamin D₃(VDR), thyroid hormone (T₃R), retinoic acid (RAR), or other members ofthe nuclear receptor superfamily. These data indicate that the LBDs ofFAR and RXRα associate in a highly specific fashion.

The only combination resulting in significant activation wasGAL4-FAR+VP-RXR. As one would expect (based on previous in vitro studies(see Hallenbeck et al., supra and Zhang et al, supra)), VP-PPAR, VP-VDR,VP-T₃R and VP-PPAR interacted productively with GAL4-RXR, therebyconfirming that these VP16 chimeras are functionally expressed.

EXAMPLE 3 Binding of FAR-RXR Complexes to DNA

It was next sought to determine the DNA binding properties of theFAR-RXRα complex. Because FAR and EcR share 100% sequence identity inthe DNA recognition helix (P-box, Cys¹⁴¹-Lys¹⁴⁵), it was examinedwhether the FAR-RXRα complex could recognize the hsp27 element responseelement (EcRE; Yao et al., Cell 71:63-72 (1992)). Electrophoreticmobility shift analysis was performed using [³²P]-labeled DNA and invitro translated FAR and RXRα. Proteins used in electrophoretic mobilityshift assays were prepared by translation in a rabbit reticulocytelysate system (TNT, Promega). Proteins (1 μl) were incubated for 20minutes at room temperature with 100,000 cpm of Klenow-labeled probes in10 mM Tris pH 8, 100 mM KCl, 6% glycerol, 0.05% NP-40, 1 mM DTT, 100ng/μl poly dI-dC and then electrophoresed through a 5% polyacrylamidegel in 0.5×TBE. The gel was autoradiographed for 1.5 hours with anintensifying screen.

Neither FR nor RXRα alone were capable of high affinity binding to thehsp27-EcRE. However, when mixed, the two proteins bound cooperatively tothe hsp27-EcRE (GGTTCA A TGCACT; SEQ ID NO:4). Binding to this elementis specific as indicated by the inability of the FAR-RXRα complex torecognize a mutated 11N-hsp27-EcRE (EcRE_(m);CGTTCA A TGCACA; SEQ IDNO:5).

The hsp27-EcRE consists of two imperfect core binding sites arranged asinverted repeats separated by 1 nucleotide (IR1; SEQ ID NO:4).Accordingly, the binding of FAR-RXRα was further examined on anidealized IR1 containing two consensus half-sites (AGGTCA A TGACCT; SEQID NO:6). The FAR-RXRα complex was also found to bind cooperatively tothe idealized IR1, but not to a mutant IR1 containing substitutionswithin the half-sites (IR1_(m); AGAACA A TGTTCT; SEQ ID NO:7). Thus,FAR-RXRα binds to ecdysone-like IR1 response elements, and representsthe first vertebrate receptor complex to possess this property.

EXAMPLE 4 Activation by Farnesoids

It was next sought to determine whether FAR possessed transcriptionalactivity that could be hormonally controlled. Based on theidentification of an EcRE as a DNA target, a reporter plasmid wasconstructed containing 5 copies of the hsp27 response element linked toa truncated mouse mammary tumor virus promoter (Yao et al., Nature366:476-479 (1993)). This reporter was cotransfected into CV-1 cellsalone, or with expression vectors for FAR and/or RXRα. Cotransfectedcells were treated with a variety of potential ligands and monitored forchanges in luciferase activity.

Transient transfections were performed as described in Example 3 usingreporter constructs (300-1000 ng/10⁵ cells), cytomegalovirus drivenreceptor (50 ng/10⁵ cells) and β-galactosidase expression vectors (500ng/10⁵ cells) as indicated in FIGS. 5A, 5B and 5C.

Thus, with reference to FIG. 5A, CV-1 cells were transiently transfectedwith hsp27-EcRE×5 MTV-luciferase alone (−) or with expression vectorsfor rat FAR and/or human RXRα. Reporter activity was assayed aftertreating cells with or without 50 μM JH III. FIG. 5A illustrates that JHIII elicited a dramatic induction (10-fold) of luciferase activity incells expressing both FAR and RXRα, relative to cells expressing eitherFAR or RXRα alone. It is of note that JH III failed to activate FAR-RXRcomplexes using the parental MTV reporter construct, which lacked theEcREs.

In contrast to the demonstrated ability of JH III to activate FAR-RXRcomplexes (see FIG. 5A), JH III fails to activate other nuclearreceptors other than FAR, as shown in FIG. 5B. Thus, the activity of thefollowing receptor/luciferase reporter pairs were assayed in thepresence of 50 μM JH III or the indicated receptor-specific ligand:Drosophila G-EcR+USP/hsp27-EcRE×5 MTV; human RXRα/CRBPII-TK; humanT₃Rβ/TREp×2-TK; and human RARα/DR5×2-TK.

As seen in FIG. 5C, the FAR-RXR complex is synergistically activated byJH III and LG69 (i.e.,(4-{1-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-napthalenyl)-1-propenyl}benzoic acid). CV-1 cells were transiently transfected as describedabove with reference to FIG. 5A, but treated with or without 50 μM JHIII, 100 nM LG69 and JH III+LG69.

Unexpectedly, JH III (50 μM) elicited a dramatic induction (10-fold) ofluciferase activity in cells expressing both FAR and RXRα (FIG. 5A).Other potential ligands including steroids, retroretinoids, eicosanoidsand bile acids had no effect. JH III appears to be specific for theFAR-RXRα complex since it failed to activate the ecdysone (EcR+USP),9-cis retinoic acid (RXR), thyroid hormone (T₃R) or all-trans retinoicacid receptors (RAR) (FIG. 5B).

Although JH III activates FR-RXRα, it fails to activate either FR orRXRα alone (FIGS. 5A and 5B). This is similar to observations with theDrosophila EcR, which requires formation of an EcR/USP or EcR/RXRcomplex for transcriptional activity (see, for example, Yao et al., inNature 366:476-479 (1993); Yao et al., in Cell 71:63-72 (1992); andThomas et al., in Nature 362:471-475 (1993)). EcR itself bindsecdysteroids with low affinity (Yao et al., (1993), supra; high affinitybinding and subsequent transcriptional activation requires coexpressionof EcR with RXR or USP. Thus, while the EcR/RXR-USP heterodimer is thephysiologically active complex, the ability to respond to ecdysone isdetermined by the EcR component of the complex. Since the EcR-RXRheterodimer is composed of two functional receptors, the complex can beactivated independently by ecdysteroids or 9-cis retinoic acid, andsynergistically by both ligands (Kensler et al., in Cancer Res.38:2896-2899 (1978)).

The structural and functional similarities between EcR and FAR promptedan examination of whether the FAR-RXRα complex could also besynergistically activated by JH III and an RXR-specific ligand (such asLG69; see Kurokawa et al., in Nature 371:528-531 (1994)). Thus, usingthe hsp27 EcRE reporter, the FAR-RXRα complex was activated 17-fold by50 μM JH III, 76-fold by 100 nM LG69 and 212-fold by the combination ofJH III and LG69. This synergistic activity required coexpression of FARwith RXRα, RXRβ or RXRλ. The ability of JH III to synergize withsaturating doses of LG69 or 9-cis RA suggests that these two compoundshave distinct targets within the FAR-RXR complex. Since LG69 haspreviously been shown to be an RXR-specific ligand, these results implythat JH III responsiveness is determined by the FAR component of theFAR-RXR complex.

EXAMPLE 5 Evaluation of Mevalonate Metabolites as FAR Ligands

JH III (cis-10,11-epoxy-3,7,11-trimethyl-trans-trans-2,6-dodecadienoicacid methyl ester) is a metabolic derivative of farnesyl pyrophosphate(FPP; 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol-pyrophosphate (see FIG.6A). FPP is derived from the mevalonate biosynthetic pathway and isitself a precursor in the synthesis of other biologically activecompounds (see FIG. 1, and Goldstein and Brown, in Nature 343:425-430(1990). Accordingly, it was decided to test whether metabolites derivedfrom the mevalonate pathway in mammalian cells could also serve asactivators of the FAR-RXRα complex.

Mevalonate can be synthesized de novo from acetyl CoA and is metabolizedinto farnesyl pyrophosphate (FPP), the metabolically active form offarnesol. FPP serves as a key intermediate in that it represents acritical branch point in the mevalonate pathway. Accordingly,metabolites of FPP contribute to a number of essential cellularprocesses. The results presented herein indicate that the FAR-RXRnuclear receptor complex responds most efficiently to farnesol andjuvenile hormone III. These findings suggest that metabolicintermediates are capable of serving as transcriptional regulators inanimal cells. Based on the results presented herein, it is likely thatthe FAR-RXR complex plays a central role in a feedback loop that servesto regulate the synthesis of enzymes within the mevalonate pathway.

Thus, CV-1 cells were transiently transfected with expression vectorsfor rat FR and human RXRα, as described above in Example 3. Cells weretreated with 50 μM concentrations of farnesol and/or farnesolmetabolites. Data is plotted in FIG. 6A as fold activation relative tountreated cells. Similar results were obtained with all-trans retinoicacid and mixed isomers of farnesol and farnesoic acid.

FIG. 6B presents a dose-response profile for the two most effectiveactivators observed in the evaluation described in FIG. 6A, i.e., JH IIIand farnesol. The experiments were performed as described above for FIG.6A, with the concentration of JH III and farnesol (mixed isomers)indicated in the Figure. Activation required concentrations in the rangeof 5-50 μM.

Remarkably, farnesol (trans-trans or mixed isomers, 50 μM) was observedto be a strong activator of FAR-RXRα (see FIG. 6A), whereas otherfarnesoids, such as farnesal, farnesyl acetate and geranylgeraniol,possessed weaker activity. In contrast, little or no activation was seenwith 50 μM concentrations of geraniol, farnesoic acid, squalene,methoprene, mevalonate, squalene epoxide, squalene dioxide, lanosterol,24,25-epoxycholesterol, pregnenolone, dehydroepiandrosterone, bile acidsor 10 μM 25-hydroxycholesterol. Mevalonate (200 μM) displayed weakactivity, provided cells were cotransfected with a mevalonatetransporter protein (see Kim et al., in J. Biol. Chem. 267:23223-23121(1992)).

EXAMPLE 6 Expression of FAR mRNA

One expectation of an intracellular metabolic activator is that it wouldbe synthesized in the same tissues where its receptor is expressed.Accordingly, the expression of FAR in rat tissues was examined byNorthern blot analysis. For Northern analysis, polyA⁺ RNA (10 μg) fromvarious rat tissues was electrophoresed through a 1% agarose gel underdenaturing conditions and transferred to a filter. The filter washybridized to the mouse FAR truncated cDNA that was [³²P]-labeled by therandom primer method (see Mangelsdorf et al., Genes Dev. 6:329-344(1992); 5×10³ cpm/μg). This probe corresponds to rat FAR sequencesspanning amino acids 1-297 which encode the N-terminus, the DNA bindingdomain (DBD) and a portion of the ligand binding domain (LBD) of FR.

Hybridization was performed overnight at 65° C. in 500 mM sodiumphosphate (dibasic:monobasic, 7:3), 1 mM ethylenediaminetetraaceticacid, 1% bovine serum albumin and 7% sodium dodecyl sulfate. The filterwas washed twice in 2×SSC (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate)at room temperature, twice in 1×SSC at 55° C. and then autoradiographedwith an intensifying screen at −70° C. for 5 days. In situhybridizations were performed as described by Bradley et al., in Proc.Natl. Acad. Sci. USA 91:439-443 (1994). Sections were apposed to KodakX-OMAT film for 10 days, and then coated with nuclear emulsion andexposed for 16 weeks.

A single transcript of 2.3 kb was observed only in liver and kidney. Nosignificant expression was detected in the brain, heart, lung, skeletalmuscle, pancreas, skin, spleen or testis.

In situ hybridization/histochemistry was performed to further localizesites of FAR expression. Antisense cRNA probes from truncated mouse FARcDNA or full-length mouse RXRβ cDNA were used. The control was atruncated rat glucocorticoid receptor sense cRNA probe. The controlprobe revealed near-background hybridization.

FAR transcripts were restricted to the liver, kidney and gut of ratembryonic day 19.5 (E19.5) embryo sections. Near background levels wereseen in other tissues and in experiments using a control probe. As onemight expect (see Mangelsdorf et al., in Genes Dev. 6:329-344 (1992)),mRNA for the heterodimerizing partner RXRβ is also found in the liver,kidney and gut, as well as other embryonic tissues. FAR expression inthe gut is prominent in the intestinal villi. In the E19.5 kidney,expression is heterogeneous, with highest FAR levels confined to therenal tubules. In the adult kidney, high levels of expression of FAR areseen in areas rich in renal tubules: the medullary rays and medullarystripe. FAR expression is also detected in the adrenal cortex of theadult mouse. Thus, FAR expression is restricted to the liver, gut,adrenal gland and kidney: tissues known to have significant flux throughthe mevalonate pathway (see, for example, Edmond et al., in Science193:154-156 (1976); Righetti et al., in J. Biol. Chem. 251:2716-2721(1976); and Wiley et al., in J. Biol. Chem. 252:548-554 (1977)).

EXAMPLE 7 FAR Gene Family

The chromosomal location of mouse FAR was determined by analysis of 2multilocus genetic crosses for inheritance of polymorphic FAR genefragments (see Danciger et al., in Mouse Genome 91:320-322 (1993), andSunada et al., in J. Biol. Chem. 269:13729-13732 (1994)).

Thus, truncated mouse FAR cDNA was used as a probe to analyze 2multilocus genetic crosses for inheritance of polymorphic Fxr genefragments: (NFS/N or C58/J×M. m musculus and (NFS/N×M. spretus)×Mspretus or C58/J. DNA from the progeny of these crosses have been typedfor approximately 700 markers including the Chr 10 markers Pfp (poreforming protein), Tra1 (tumor rejection antigen gp96), Ifg (interferonγ), Gli (glioma associated oncogene) and Gad1-ps1 (glutamic aciddecarboxylase 1 pseudogene).

To the right of the map (FIG. 7) are the recombination fractions betweenadjacent loci; percent recombination and standard errors are shown inparentheses. Human map locations for the homologues of individual genesare indicated to the left of the map.

To determine whether there may be related genes that comprise a FR genefamily, Southern blot analysis of rat genomic DNA was performed and thepatterns obtained under high and low stringency hybridization werecompared. Thus, duplicate samples of Lewis rat DNA (10 μg) were digestedwith a variety of restriction enzymes and electrophoresed through a 1%agarose gel. DNA was digested with restriction enzyme, transferred to anitrocellulose filter and then hybridized with the [³²P]-labeled mouseFAR truncated cDNA probe under high or low stringency conditions. As onemight expect, high stringency conditions revealed a limited number ofspecific bands for each restriction enzyme. Under low stringencyconditions, many additional bands were obtained, suggesting theexistence of one or more FAR-related genes in the rat genome. Althoughfurther analysis is required to determine whether these relatedsequences are functionally expressed, these findings raise thepossibility that additional farnesoid activated receptors will beidentified.

Southern analysis revealed HindIII digested fragments of 7.5 kb, 6.0 kband 3.0 kb in NFS/N mouse DNA and 25.0, 7.5 and 3.0 kb in M. spretus.ScaI digestion produced fragments of 23.1 kb in NFS/N and 28 kb in M.m.musculus. The inheritance of these fragments demonstrated that Fxr,the gene encoding FR, is localized near the Tra1 locus on mouseChromosome 10 (FIG. 7). This map location is within a region ofconserved linkage with human chromosome 12q suggesting a possible maplocation for human Fxr.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. A method for modulating gene expression mediated by farnesoidactivated receptor (FAR) polypeptides in isolated cells containing FARpolypeptides, said method comprising subjecting said cells to a changein the concentration of at least one farnesoid, thereby modulating saidgene expression.
 2. A method according to claim 1 wherein said FARpolypeptide is characterized by reference to the unique tissuedistribution thereof, wherein expression of FAR polypeptides isrestricted to the liver, gut, adrenal gland and kidney.
 3. A methodaccording to claim 1 wherein said FAR polypeptide is characterized by:(1) being responsive to the presence of farnesoid(s) to regulate thetranscription of associated gene(s) having response elements organizedas an IR1; (2) having a relative molecular mass of about 54,000; and (3)having a DNA binding domain of about 66 amino acids with 9 Cys residues,wherein said DNA binding domain is encoded by a nucleic acid sequencethat hybridizes under low stringency conditions of 42° C. for 24 hourswith 0 or 10% formamide, 1×Denhardt's solution, 6×NET 0.2% SDS, and 100μg/ml denatured salmon sperm DNA, and washed four times for 20 minutesat 25° C. in 2×SSC, 0.1% SDS to a nucleic acid molecule encoding aminoacid residues 124-189 set forth in SEQ ID NO:2.
 4. A method according toclaim 3 wherein said FAR polypeptide is further characterized by: (4)having a ligand binding domain encoded by a nucleic acid sequence thathybridizes under low stringency conditions of 42° C. for 24 hours with 0or 10% formamide, 1×Denhardt's solution, 6×NET 0.2% SDS, and 100 μg/mldenatured salmon sperm DNA, and washed four times for 20 minutes at 25°C. in 2×SSC, 0.1% SDS to a nucleic acid molecule encoding amino acidresidues 250-469 set forth in SEQ ID NO:2.
 5. A method according toclaim 1, wherein said FAR polypeptide is encoded by a nucleic acidsequence that hybridizes under low stringency conditions of 42° C. for24 hours with 0 or 10% formamide, 1×Denhardt's solution, 6×NET 0.2% SDS,and 100 μg/ml denatured salmon sperm DNA, and washed four times for 20minutes at 25° C. in 2×SSC, 0.1% SDS to a DNA encoding the amino acidsequence shown in SEQ ID NO:2.
 6. A method according to claim 1, whereinsaid FAR polypeptide has the same amino acid sequence as that shown inSEQ ID NO:2.
 7. A method according to claim 1 wherein said farnesoid hasthe structure:

wherein each R is independently lower alkyl or alkoxy, each R′ isindependently selected from hydrogen, lower alkyl or alkoxy, each R″ isindependently selected from hydrogen, lower alkyl or alkoxy, X isselected from —CH₂OH, —CH₂OAc, —CO₂H, or —CO₂Me, n is 2 or 3, each q isindependently 1 or 2, each q′ is independently 1 or 2, and q and q′ arethe same.
 8. A method according to claim 7 wherein each R is methyl,each R′ is hydrogen, each R″ is hydrogen, X is —CH₂OH, n is 2, and eachq and q′ is
 1. 9. A method according to claim 7 wherein each R ismethyl, each R′ is hydrogen, each R″ is hydrogen, X is —CO₂H, n is 2,and each q and q′ is
 1. 10. A method according to claim 7 wherein thepolyene backbone contains an epoxide functionality, each R is methyl,each R′ is hydrogen, each R″ is hydrogen, X is —CO₂Me, n is 2, and eachq and q′ is
 1. 11. A method according to claim 7 wherein each R ismethyl, each R′ is hydrogen, each R″ is hydrogen, X is —OAc, n is 2, andeach q and q′ is
 1. 12. A method according to claim 7 wherein each R ismethyl, each R′ is hydrogen, each R″ is hydrogen, X is —CH₂OH, n is 3,and each q and q′ is 1.